Clostridium difficile is the causative agent of pseudomembranous colitis (PMC) and appears to be endemic in some long-term care facilities. C. difficile produces at lest two toxins, toxin A, an atypical enterotoxin and toxin B, a potent cytotoxin. Little is known of the mode of action of toxin B or structure/function relationships, or its contribution to the development of the disease. The specific aims of the proposed research are: (1) clone and sequence the toxin B gene, (2) to identify the receptor binding domain of the toxin and (3) to identify the catalytic portion of the molecule. These goals are part of long term objectives to eventually determine the mode of action of both toxins, to completely define their functional domains, to elucidate the mechanism of cellular uptake and processing, and examine the role of each toxin in the disease process. In preliminary work highly purified toxin B has been prepared and putative clones of toxin B have been isolated. The clones were identified using affinity purified antibody directed against toxin B. These clones are presently under analysis. DNA sequencing of the putative clones will be achieved using dideoxy sequencing methods. The delineation of the receptor binding and catalytic regions of toxin B will be accomplished by the combined use of in vitro mutagenesis of the cloned toxin gene and the generation of proteolytic derivatives from the native toxin molecule. Receptor binding function will be evaluated by competition assays with radiolabelled native toxin. Identification of the catalytically active portion of the molecule will be accomplished by the direct introduction of toxin derivatives into the cytoplasm of the target cell. This will be done by loading erythrocyte ghost with the toxin B derivative and fusing the loaded erythrocyte ghosts with target cells via viral hemagglutinin driven fusion.